Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Müntefering, Thomas; Michels, Alexander; Pfeuffer, Steffen; Meuth, Sven G.; Ruck, Tobias

doi:10.3791/58901

Cited by 5 publications

(8 citation statements)

References 5 publications

(6 reference statements)

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“…CD31 + cells retained in the LS column were eluted with PEB buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and centrifuged at 300 g for 5 minutes. CD45 – CD31 + cells corresponding to endothelial cells were directly pelleted for RNA or protein isolation ( 71 – 73 ).…”

Section: Methodsmentioning

confidence: 99%

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Bartoli¹,

Debant²,

Chuntharpursat‐Bon³

et al. 2022

Journal of Clinical Investigation

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Piezo1 forms mechanically-activated non-selective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial apoptosis, with no effect on thrombospondin-1 (TSP1), a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without effect on Tsp1. In the endothelial cells, Piezo1 was required for normal expression of endothelial nitric oxide synthase (eNOS). The data suggest an endothelial-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.

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Section: Methodsmentioning

confidence: 99%

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Bartoli¹,

Debant²,

Chuntharpursat‐Bon³

et al. 2022

Journal of Clinical Investigation

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“…We do acknowledge several other published protocols regarding (endothelial) cell isolation from murine organs (e.g. brain: ( Assmann et al., 2017 ; Czupalla et al., 2018 ; Ruck et al., 2014 ); muscle: ( Müntefering et al., 2019 ); lung: ( Cheung and Marelli-Berg, 2018 ; Nakano et al., 2018 )). We would like to point out that our protocols' advantages and novelty mostly rely on the combination of a rapid method for fresh cell isolation, simplicity of the protocols, specific design per organ to get highest quantity of ECs and high purity and quality of cells directly after the isolation (see Figures 5 and 6 ).…”

Section: Limitationsmentioning

confidence: 99%

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle

et al. 2021

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Summary Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020) , Rohlenova et al. (2020) , and Goveia et al. (2020) .

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“…However, in our specific case, magnetic-activated cell sorting (MACS) offered advantages compared to FACS such as a lower device cost, higher throughput and faster processing time ( Tomlinson et al, 2013 ; Sutermaster and Darling 2019 ; Pan and Wan 2020 ). The most recent work on the isolation of primary murine SkMVECs was published by Müntefering et al (2019) . There, a protocol was established yielding CD31-positive murine SkMVECs with a purity of up to 95% using MACS ( Müntefering et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

“…The most recent work on the isolation of primary murine SkMVECs was published by Müntefering et al (2019) . There, a protocol was established yielding CD31-positive murine SkMVECs with a purity of up to 95% using MACS ( Müntefering et al, 2019 ). Taken together, only a few protocols have been published describing the isolation of primary SkMVECs and none of those has been applied to human tissue.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, for co-culturing approaches in skeletal muscle tissue engineering, a protocol on the combined isolation of both primary myoblasts and primary SkMVECs from the same biopsy is not existing. Therefore, originally based on the protocol of Müntefering et al (2019) , we developed a protocol for the co-isolation of SkMVECs and satellite cell-derived myoblasts from the same human skeletal muscle tissue biopsy.…”

Section: Introductionmentioning

confidence: 99%

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Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle

Wüst

Terrie

Müntefering

et al. 2022

Front. Bioeng. Biotechnol.

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Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.

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Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Cited by 5 publications

References 5 publications

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle

Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle

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